(1) Field of the Invention
This invention relates to a method and apparatus for disrupting spores to aid subsequent analysis, especially to a method and apparatus for fast release of intrasporal DNA.
(2) Description of the Art
There is a growing need to be able to detect and identify spore forming bacteria. For example in the food industry there is a desire for rapid analysis of food stuffs to detect the presence of any bacterial spores, such as Bacillus cereus, before they can germinate and spoil produce and/or cause illness. Equally there is a need for rapid detection/identification of spores such as Bacillus anthracis. 
The current gold standard for detection of spores is germination via heat activation and outgrowth. However this process takes up to 48 hours and requires skilled personnel and therefore is unsuitable for rapid identification.
More rapid tests exploit antibodies associated to the surface of the spores (exosporium) for detection. Handheld immunochromatographic tests are available but the sensitivity of such test are low.
Matrix Assisted Laser Desorption and Ionisation (MALDI) is a standard technique for transferring large biomolecules into the vapour phase for mass spectrometric analysis. MALDI has been used to detect specific biomarkers associated with the outer layers of the spores.
In a MALDI analysis the analyte of interest is mixed with a suitable matrix material and a solvent on a substrate. The solvent is then evaporated to leave the analyte co-crystallised with the matrix material. A pulsed UV laser source is then directed to irradiate the sample. The matrix material absorbs the laser light and a rapid temperature increase causes disintegration of the matrix ejecting a plume of sample. The ejection plume is input to a mass spectrometer to analyse the ionised biomolecules and hence the irradiation step is performed in a high vacuum.
The various biomarkers associated with the exosporium can then be identified. However the method is not good at discriminating between different Bacillus species as the origin and identity of the biomarkers may be unclear and different species may have similar biomarkers. To increase the number of released biomarkers corona plasma discharge may be used or sonication pre-treatments could be used but discrimination between species is still relatively poor.
Sonication may also be used to modify the surface of spores so as to aid subsequent detection in an immunoassay, for instance immunoassays involve the binding of an analyte to a specific antibody contained on the surface of a sensor. Detection sensitivity can be improved by modification of the surface of the species to be detected so as to improve subsequent binding to the antibodies on the biosensor.
Another method of screening for spores is to completely disrupt the spore so as to release intrasporal DNA for subsequent analysis via polymerase chain reaction (PCR) assays. For instance ultrasonication has recently been proposed to completely disrupt spores in ‘Belgrader P.; Hansford D.; Kovacs G. T. A.; Venkateswaran, K; Mariella, R.; Milanovich, F.; Nasarabadi, s.; Okuzumi, m; Pourahmadi, F.; Northrup, M. A. Analytical Chemistry 1999, 71, 4232-4236’. However the samples can require pretreatments of up, to 90 minutes and so far the amount of intracellular DNA released has been low so the technique would not currently be sensitive enough for most applications.